Historically, treatment with estrogens was commonly used to treat estrogen receptor (ER)-positive breast cancer. Estrogen therapy fell out of favor upon the introduction of the anti-estrogen tamoxifen, which showed similar efficacy with fewer adverse effects. More effective anti-estrogens have since been developed (e.g., aromatase inhibitors, fulvestrant), but anti-estrogen resistance remains a common problem. Approximately 1/3 of women diagnosed with early-stage ER+ breast cancer will ultimately experience cancer recurrence that becomes progressively resistant to all approved anti-estrogens. Estrogen therapy has been explored as a treatment option for patients with advanced anti-estrogen resistant disease, and clinical trials demonstrate that ~30% of patients experience clinical benefit. Despite the proven success of estrogen therapy, clinical use is limited; the success of anti-estrogens has led to the prevailing view that estrogens feed tumor growth, making estrogen therapy an unpopular treatment option. Additionally, the molecular mechanism underlying tumor response and a biomarker to predict response to estrogen therapy are unclear, leading to difficulty in identifying patients likely to benefit from this treatment. Mechanistic understanding has been limited to studies in two monoclonal derivatives from one ER+ breast cancer cell line. We have assembled a panel of 3 tumor models and 2 cell line models of response to estrogen therapy to comprehensively study the molecular mechanism of response. Preclinical and clinical data suggest that high levels of ER expression are associated with therapeutic response to estrogens. Preliminary clinical data suggest that somatic mutations in the ligand- binding domain of ER, which promote constitutive activation, may modulate therapeutic response to the natural estrogen 17b-estradiol (E2). Preliminary findings also suggest that treatment with E2 induces ER-dependent DNA damage in models of response to estrogen therapy. This indicates that stimulation of high levels of ER transcriptional activity leads to the induction of DNA damage, and that this incurred DNA damage may underlie therapeutic response to estrogen. Specific Aim 1 will use cell line and tumor models to determine how high levels of ER activity induce DNA damage, and whether this leads to cell cycle arrest and apoptosis. Additionally, Specific Aim 1 will test whether therapeutic response to E2 can be enhanced by combination treatment with drugs inhibiting DNA repair and response to DNA damage. Specific Aim 2 will test whether commonly occurring mutations in the ER ligand-binding domain modulate therapeutic response to E2 treatment. Findings from these studies will expand the clinical use of estrogens as a treatment option for advanced, anti-estrogen resistant ER+ breast cancer, and may lead to novel strategies to enhance response.